AMPAR and spine dysfunction or dysregulation underlies many CNS diseases including depression, autism, PTSD, epilepsy, and stroke-induced neuronal damage. Precise postsynaptic localization of AMPARs is critical for fast synaptic transmission. It depends on PSD-95 and its interaction with auxiliary AMPAR subunits called TARPs. Despite its central role in targeting AMPARs, it is unknown how PSD-95 itself is anchored at the postsynapse. Our preliminary data suggest that A) ?-actinin binds to the first 13 residues of the N-terminus of PSD-95; B) knock-down (KD) of ?-actinin reduces postsynaptic PSD-95 content and mEPSCs, the latter phenocopying KD of PSD-95; C) mutating either Lys10 or Lys11 to Glu (K10E, K11E) specifically impairs PSD- 95 binding to ?-actinin and postsynaptic targeting of PSD-95 and of AMPARs; D) peptide PSD95(1-13) displaces PSD-95 from ?-actinin; E) injection of PSD95(1-13) decreases mEPSC amplitude. We hypothesize that ?-actinin is critical for postsynaptic anchoring of PSD-95 and thereby AMPAR-TARP complexes. Proving this hypothesis will fundamentally advance our understanding of postsynaptic AMPAR localization. A), B), and D) are final data. Aims 1 and 2 will further scrutinize C) and E), i.e., whether mutating K10E and K11E or injecting PSD95(1-13) affect synaptic PSD-95 and AMPAR taregting using fluorescence microscopy and mEPSC. NMR structural analysis will identify residues in ?-actinin that are important for PSD-95 binding. KD of endogenous ?-actinin and replacement with either WT or mutant ?-actinin will show whether mutant ?-actinin is not able to rescue the KD effect on PSD-95, in contrast to our rescue with WT ?-actinin. Aim 3 is to test whether NMDA-induced Ca2+ influx displaces PSD-95 from ?-actinin and thereby from postsynaptic sites along with AMPARs via calmodulin (CaM). We found that Ca2+/CaM binds to the N-terminus of PSD-95 to dislodge ?-actinin. Our structural NMR analysis of a complex between Ca2+/CaM and the first 71 aa of PSD-95 identified Y12 in PSD-95 as critical for CaM binding. Mutating Y12 to Glu (Y12E) prevents Ca2+/CaM binding without affecting ?-actinin binding or postsynaptic localization of PSD-95. NMDA-induced Ca2+ influx displaces a portion of WT but not Y12E PSD-95 from spines. In fact, Y12E exhibits a large increase rather than decrease in spines upon Ca2+ influx. We will test whether this mutation and other manipulations unmask a mechanism that leads to postsynaptic accumulation of PSD-95 and AMPARs rather than their decrease. Such a decrease is usually seen following 5 min NMDA treatment and is referred to as chemical LTD. This exciting new direction will elucidate how Ca2+ influx can cause LTD rather than LTP.